Over the past decade, we have witnessed tremendous progress in our understanding of the role of RNA molecules in the regulation of gene expression. The main contribution to this progress was offered by the discovery of RNA interference (RNAi). First identified in C. elegans by Fire and Mello [Fire, et al. 1998], RNAi is an evolutionary conserved mechanism that brings about a sequence specific, post transcriptional gene silencing (PTGS) through the use of short RNAs. The basic idea behind RNAi is that a short RNA duplex of 21-23 nucleotides, termed short interfering RNA or siRNA, complementary to a segment of the mRNA, can be exogenously synthesized and introduced into the cell. This triggers a process which finally degrades the homologous mRNA and inhibits the production of the corresponding protein. Several types of short RNAs, including short interfering RNA (siRNA), micro RNA (miRNA), tiny non-coding RNA (tncRNA), and short hairpin RNA (shRNA), may be involved in RNAi process [Novina, et al. 2004, Paddison, et al. 2008, Engelke, et al. 2005].
The major limitations for the use of siRNA both in vitro and in vivo are the instability of naked siRNA in physiological conditions, rapid clearance from the bloodstream, and the inability to cross the cellular membrane to gain access to the intracellular environment. Because of their small size and hydrophilicity, a significant portion of administrated naked siRNAs are excreted through the reticuloendothelial system (RES) [Moghimi, et al. 1999]. It was also reported that highly charged particles can be recognized by the RES more rapidly than neutral or slightly charged particles [Benoit, et al. 2006, Mahato. 2005]. Furthermore, nucleic acid (NA)-based drugs are subjected to enzymatic degradation during circulation and within the cell, resulting in insufficient drug potency at the target site. Chemical modifications in the sugars, nucleobases, and the phosphate ester backbone of siRNA have been applied to improve its nuclease resistance without interfering with the silencing efficiency [Manoharan 2004, Verma, et al. 2003, Zhang, et al. 2006]. Conjugation with hydrophobic functional groups has also enhanced the cellular uptake [Oliviera et al., 2006].
In comparison with chemical modifications of NAs, which is time-consuming and costly, carrier-mediated strategies are emerging as a simple and fast means to formulate NA therapeutics and protect them from degradation. The carriers, including viral vectors, lipids, polymers, and peptides, co-assembled or covalently conjugated with siRNA, are designed to enhance cell targeting, prolong drug circulation time, and improve membrane permeation.
Because of their diversity and versatility in design, through the use of amino acids with different physicochemical properties, peptides have been employed to deliver synthetic drugs, small molecules, bioactive peptides, therapeutic proteins, and NAs by a mechanism that has not yet been fully understood. These peptides may include protein-derived cell penetrating peptides (CPPs) [Langel 2007], cationic peptides [Benoit, et al. 2006], designed amphiphilic peptides [Oehlke, et al. 1998], fusogenic peptides [Mok, et al. 2008], cell targeting peptides (CTP) [vives 2005], and peptides containing a nuclear localization signal [Cartier, et al. 2002]. Cationic peptides rich in basic amino acids can electrostatically interact with small NAs or condense NA into small stable particles. CPPs can facilitate the translocation of the complex through the cell membrane. Histidine-rich pH-sensitive or fusogenic peptides can enhance the endosomal escape and cytoplasmic release of the gene complex. Involvement of CTPs in gene delivery systems mediates cell and/or tissue-specific targeting. Finally, attachment of a NLS peptide improves nuclear localization of the gene complex.
Among CPPs, only few have shown high transfection efficiency with low cytotoxicity and immunogenicity. Tat and Penetratin are the most widely investigated peptides among protein-derived peptides. Trans-activating transcriptional activator (Tat) from Human Immunodeficiency Virus 1 (HIV-1), discovered by Frankel and Pabo in 1988, can be efficiently taken up by several cell types in culture [Jarvert, et al. 2007].
Specific cell-penetrating peptides (CPPs) identified as effective carriers for NAs have been described, see e.g. International Patent Application Publication Nos. WO 2007/076904 to Brock et al. and WO 2007/069090 to Divita et al., although not all describe the transport of siRNA, see e.g. U.S. Pat. No. 7,163,695 to Mixson and U.S. Pat. No. 7,112,442 to Rice et al.
In many of these carrier-mediated delivery systems the NA is covalently linked to a carrier peptide of a specific sequence, see e.g. International Patent Application Publication No. WO 2008/063113 to Langel et al. and United States Patent Application Publication No. US 2005/0260756 to Troy et al. Specific peptides have been linked to NA via chemical linkers, see e.g. WO 2008/033285 to Troy et al and WO 2007/069068 to Alluis et al. U.S. Pat. No. 7,420,031 to Karas reports a peptide capable of delivering NAs to an intracellular compartment of a cell; the peptide-cargo moiety complex is formed by a chemical cross-linking or bridging method. U.S. Pat. No. 7,306,784 to Piwinica-Worms describes use of cell membrane-permeant peptide conjugate coordination and covalent complexes having target cell specificity.
United States Patent Application Publication No. US 2008/0234183 to Hallbrink et al. describes methods for predicting, designing, detecting and verifying CPPs. The CPP-cargo complexes of Hallbrink et al. involve a covalent linkage between the CPP and cargo molecule.
International Patent Application Publication No. WO 2010/039088 to Kariem et al. describes the use of some stearylated linear or branched CPPs, in particular Transportan and Penetratin, in NA delivery.
U.S. Pat. No. 6,800,481 to Holmes et al. describes the self-assembly of amphiphilic peptides, i.e., peptides with alternating hydrophobic and hydrophilic residues, into macroscopic membranes.
International Patent Application Publication No. WO 2003/106491 to Langel et al. describes methods for predicting, designing, detecting, and verifying CPPs and their use for improved cellular uptake of a cellular effector coupled to the CPP.